Introduction
The process of cell line development for the production of antibody-based therapeutics can vary widely amongst researchers. The ultimate goal is to find a clone that stably produces large quantities of a high-quality monoclonal antibody.
The measurement of IgG production is a crucial step at many stages in the development and manufacturing of monoclonal antibodies (Figure 1). Commonly used methods for IgG quantitation require either special instrumentation and skilled personnel, such as HPLC and surface interferometry, or time-consuming assays like ELISA (enzyme-linked immunosorbent assay). Although the ELISA is a well-established method for protein quantitation, it is a lengthy, multi-step process (Table 1). Here, we introduce the use of the Valita®TITER assay from Valitacell for measuring IgG titers during the antibody development and manufacturing process.
The ValitaTITER Assay measures IgG concentrations from 2.5 – 100 mg/L with a simple add-and-read protocol. The assays are performed in less than one hour and can be incorporated into the bioprocess workflow in a 96 well plate format. The assay is high throughput and can be fully automated. Analysis can be carried out in cell culture media with a low sample volume and without complex preparation steps. Assay detection can be performed on multi-mode microplate readers from Molecular Devices: SpectraMax® iD5 Multi-Mode Microplate Reader, SpectraMax® M5 Multi-Mode Microplate Reader, and SpectraMax® i3x Multi-Mode Microplate Reader with the Fluorescence Polarization Detection Cartridge.
Figure 1. The cell line development process from cell transfection to scale-up.
| ValitaTITER | Surface Interferometry | ELISA | HPLC | |
|---|---|---|---|---|
| Total assay time(96 samples) | 45 min | 55–65 min | 6+ hours | 25–45 hours |
| Measurement range(mg/L) | 2.5–100 | 0.025–2000 | 0.5–5 | >10 |
| Sample volume | 5 µL–30 µL | 180 µL + | 100 µL | 1–2 mL |
| Precision | < 2% | < 5% | < 5–10% | < 2% |
Table 1. Performance of ValitaTITER assay compared to other methods used for IgG quantitation.
Assay principle
The ValitaTITER assay is based on detection of IgG Fc interactions with Protein G using fluorescence polarization (FP) technology. Each well of a 96- well microtiter plate is coated with a fluorescently labeled IgG-binding peptide, Protein G. When samples are added to the wells, the Protein G molecules are resuspended and binding occurs. The rate of molecular motion of Protein G slows down when it is bound to antibodies, resulting in an increase of the FP value (Figure 2).
Figure 2. ValitaTITER assay principle using the FP technology.Free Protein G molecules are smaller and rotate faster in the buffer solution. As a result, the polarized excitation light loses polarization (top). When Protein G molecules bind to the antibodies in the samples, the rotation of the larger complex slows down resulting in increase of the FP values (bottom).
Materials
- ValitaTITER Assay Kit(Valitacell cat. #00010)
- IgG protein standard (Sigma cat. #I2511)
- CD CHO Medium (Thermo Fisher Scientific cat. #10743011)
- In-process samples: cell culture supernatant samples generated by recombinant IgG producing CHO cell lines cultivated in suspension
- SpectraMax iD5Multi-Mode Microplate Reader equipped with 535-nm FP emission filters
- SpectraMax i3xMulti-Mode Microplate Reader with Fluorescence Polarization (FP-FLUO) Detection Cartridge
- SpectraMax M5Multi-Mode Microplate Reader
Methods
IgG standards were serially diluted in CD CHO cell culture medium to concentrations between 2.5 and 100 mg/L. A range of IgG samples of cell culture supernatant were diluted as required in cell culture media. 60 μL of ValitaMab reconstitution buffer (culture medium can be used here instead) was pipetted into each well of the ValitaTITER plate, followed immediately with 60 μL of prepared cell samples or protein standards. Contents were mixed and incubated in the dark for 30 minutes at room temperature. FP signal was measured on the SpectraMax iD5 reader using the onboard monochromator optics for excitation (485-nm) and a 535-nm filter set for emission. Data were generated and analyzed with a preconfigured SoftMax Pro protocol (Figure 3). FP was also measured on SpectraMax i3x and SpectraMax M5 readersto verify their performance (data not shown).
Figure 3. ValitaTITER assay workflow. (A)ValitaMab reconstitution buffer is pipetted into each well of the ValitaTITER plate. (B)Samples or IgG standards are added to the wells. (C) The plate is incubated for 30 min to allow binding to occur. (D) FP is measured on the SpectraMax iD5 reader using SoftMax Pro Software.
Comparison data were generated using Protein A HPLC. Experiments were carried out according to the manufacturer’s instructions.
Results
With the ValitaTITER assay, an IgG standard curve was obtained using a simple add-and-read method with no wash steps and a small (5-30 μL) sample volume. On the SpectraMax iD5 reader, best results were obtained using built-in monochromator optics for excitation at 485-nm and a 535-nm FP filter set for emission. Standards from 3.1 to 100 mg/L were detected with a high degree of linearity (r2=0.998) across the entire range (Figure 4). Similar data were obtained with the SpectraMax i3x and SpectraMax M5 readers (data not shown). A preconfigured protocol in SoftMax Pro Software automated the mP calculations and curve plotting.
Figure 4. IgG standard curve generated using the ValitaTITER assay on the SpectraMax iD5 plate readerData were plotted using a linear curve fit in SoftMax Pro software (r2=0.998). Error bars show standard deviation of 6 replicates..
Results from the ValitaTITER assay for conditioned media samples from fed-batch reactors were compared to HPLC. The two methods showed a strong correlation, with r2=0.994 (Figure 5).
Figure 5. Comparative analysis of IgG quantitation by ValitaTITER assay and Protein A HPLC analysis.IgG titer in a range of conditioned media samples from fed-batch bioreactors was quantified by ValitaTITER fluorescence polarization assay. The samples were also analyzed by Protein A HPLC.
Conclusion
IgG quantitation must be performed at many steps in the cell line development process to ensure a high-quality final product for scale-up, so an assay that enables accurate results with a minimal investment of time is critical to success. Here we demonstrate that the IgG quantitation data obtained from the ValitaTITER assay matched those from HPLC analysis, widely regarded as a gold-standard method, but were generated in less than 3% of the time required for HPLC.
The ValitaTITER assay is a homogeneous, high-throughput method for precise and rapid quantitation of IgG in the cell line development workflow. This 96-well assay has been fully validated on the SpectraMax iD5 reader and other Molecular Devices plate readers with FP detection to ensure reliable results. SoftMax Pro Software minimizes setup time for detection and automates standard curve fitting and sample quantitation.
简介
在不同的研究人员中，以抗体为基础的治 疗药物的细胞系开发过程可能有很大不同。 最终目标是找到可以稳定产生大量单克隆 抗体的细胞克隆。
IgG 产物检测在开发和生产单克隆抗体的 多个阶段都是非常重要的一步 ( 图 1 )。通 常 IgG 定量的方法都需要专门的仪器和技 术人员，例如 HPLC 和表面干涉测量，或 是要花费大量时间的 ELISA 检测 ( 酶联免 疫吸附实验 )。尽管 ELISA 是蛋白定量的 成熟方法，但它也是一种冗长的、多个步 骤的实验过程 ( 表 1 )。这里，我们向大家 介绍使用 Valitacell 公司的 Valita®TITER 实验来检测抗体开发和生产过程中 IgG 的 方法。
ValitaTITER 实验使用简单“add-and-read” ( 添加和读数 ) 方式测定 2.5 ‒ 100 mg/L 浓 度的 IgG。此实验过程不到 1 小时即可完 成且能够整合到以 96 孔板为规格的生物 反应工作流程中。此实验可实现高通量和 全自动。其过程可在细胞培养基中用少量 样品进行，没有复杂的准备步骤。可用于实验检测的 Molecular Devices 公 司多功能微孔读板机有：SpectraMax® iD5多功能微孔读板机、带荧光偏振检测 卡盒的 SpectraMax® i3x 多功能微孔读板 机和 SpectraMax® M5 多功能微孔读板机。
图 1 从细胞转染到扩大生产的细胞系开发过程
| ValitaTITER | 表面干涉测量 | ELISA | HPLC | |
|---|---|---|---|---|
| 总实验时间 ( 96 个样品 ) | 45 min | 55–65 min | 6+ hours | 25–45 hours |
| 测定范围 ( mg/L ) | 2.5–100 | 0.025–2000 | 0.5–5 | >10 |
| 样品体积 | 5 µL–30 µL | 180 µL + | 100 µL | 1–2 mL |
| 精确度 | < 2% | < 5% | < 5–10% | < 2% |
表 1 比较 ValitaTITER 实验与其他方法在 IgG 定量检测方面的性能表现
实验原理
ValitaTITER 实验基于对 IgG Fc 片段与 蛋白 G 的相互作用采用荧光偏振 ( FP ) 技术检测。96 孔微滴度板的每个孔中 都包被有荧光标记的 IgG 结合多肽， 蛋白 G。当加入样品到孔板中时，蛋白 G 分子被重悬并发生结合。一旦结合抗 体，蛋白 G 分子的运动速率便会下降， 导致荧光偏振 ( FP ) 值的增加 ( 图 2 )。
图 2 ValitaTITER 实验原理使用 FP 技术检测。自由的蛋白 G 分子很小且在缓冲液中快速旋转。结果 是偏振激发光丢失了偏振性 ( 上图 )。当蛋白 G 分子在样品中结合了抗体，较大的复合体旋转就会减 慢导致 FP 值的增加 ( 下图 )
材料
- ValitaTITER 检测试剂盒 (Valitacell cat. #00010)
- IgG 蛋白标准品 (Sigma cat. #I2511)
- CD CHO 培养基 (Thermo Fisher Scientific cat. # 10743011)
- 实验过程中的样品：悬液培养产生 CHO 细胞系的重组 IgG 培养上清液
- 安装有 535-nm FP 发射滤片的 SpectraMax iD5 多功能微孔读板机
- 带有荧光偏振 ( FP-FLUO ) 检测卡盒 的 SpectraMax i3x 多功能微孔读板机
- SpectraMax M5 多功能微孔读板机
方法
IgG 标准品梯度稀释到 CD CHO 细胞培 养基中使浓度在 2.5 和 100 mg/L 之间。 细胞培养上清中的一系列 IgG 样品按 需要稀释到细胞培养基中。加入 60 µL ValitaMab 重组缓冲液到 ValitaTITER 板的每个孔中，然后再迅速加入 60 µL 准备好的细胞样品或蛋白标准品。混 匀样品或标准品并在室温下避光孵育 30 分钟。在 SpectraMax iD5 读板机 上使用内置的光栅光路激发并用 535-nm 发射滤片检测 FP 信号。使用 SoftMax Pro 软件中的预设模板获取数 据并进行分析 ( 图 3 )。同样在SpectraMax i3x 和 SpectraMax M5 读板机上 检测 FP 以验证它们的性能表现 ( 数据 未示 )。
图 3 ValitaTITER 实验流程。 (A)ValitaMab 重组缓冲液加入到 ValitaTITER 板的每个孔中。 (B)样品 或 IgG 标准品加入到孔中。 (C) 孔板孵育 30 分钟为结合的发生。(D) 使用 SoftMax Pro 软件在 SpectraMax iD5 读板机上检测 FP
用蛋白 A 的 HPLC 数据作对比。根据 生产厂家操作说明进行实验。
结果
ValitaTITER 实验利用简单的“add-andread”( 添加和读数 ) 方式获得 IgG 标 准曲线，中间无洗涤步骤且只需少量 样品 ( 5-30 µL )。在 SpectraMax iD5 读板机上使用内置的光栅激发光路和 535-nm FP 发射滤片可获得最佳结果。 浓度从 3.1 到 100 mg/L 的标准品所检 测到的结果在整个浓度范围内均保持 了高度线性 (r2=0.998) ( 图 4 )。SpectraMax i3x 和 SpectraMax M5 读板机也 获得了相似的数据 ( 数据未展示 )。 SoftMax Pro 软件中的预设模板可自动 计算出 mP 结果并绘制曲线图。
图 4 使用 ValitaTITER 实验在 SpectraMax iD5 读板机上生成 IgG 标准曲线。数据采用 SoftMax Pro 软件中的线性拟合绘制曲线图 (r2=0.998)。误差线表示 6 个重复标品的标准偏 差
对进料间歇反应器条件培养基样品的 ValitaTITER 实验结果与 HPLC 进行了 比较。两种方法呈现出高度的一致性， r2=0.994 ( 图 5 )。
图 5 ValitaTITER 实验的 IgG 定量结果和蛋白 A 的 HPLC 分析结果进行比较。。进料间歇生 物反应器中一系列条件培养基的 IgG 滴度通过 ValitaTITER 荧光偏振实验进行定量。这些样 品同样也通过蛋白 A 的 HPLC 进行分析
总结
IgG 定量必须在细胞系开发过程的多个 步骤中进行以保证最终扩大化生产的产 品质量，因此一种以尽可能少的时间 获得最准确结果的测定方法对成功至 关重要。这里我们展示了由 ValitaTITER 实验得到的 IgG 定量数据与 HPLC 的 分析结果高度匹配，后者被广泛认为 是检测的金标准，然而前者所需的时 间还不到 HPLC 的 3% 。
ValitaTITER 实验是一种均相的、高通 量的方法，可在细胞系开发工作流程中 精确快速的检测 IgG 含量。这种实验的 96 孔板检测已在 SpectraMax iD5 读 板机和其他具有 FP 功能的 Molecular Devices 公司读板机上被充分验证通过， 保证结果的可靠性。而 SoftMax Pro 软 件则大大减少检测参数的设置时间并 自动拟合标准曲线和计算样品浓度。